Diabetes is developing in epidemic proportions worldwide, with the cost in human suffering and health care projected to grow exponentially over the coming decades. Diabetic retinopathy is among the most common and devastating complication of diabetes. Clinical and experimental observations indicate that interactions between endothelial cells and pericytes, the two cell types that form capillaries, are central to the health and function of the retinal vasculature. The formation of the microvasculature is a complex process that involves a number of growth factors as well as homotypic and heterotypic cell-cell interactions; however, the mechanism that underlies long-term capillary stabilization is not known. The Notch pathway is a fundamental cell signaling mechanism linking the developmental fate of one cell to that of its neighbor. Studies in transgenic mouse models and humans carrying mutations in Notch pathway have demonstrated that vascular development is exquisitely sensitive to the dosage of Notch signals. Specifically, Notch appears to play a central role at the later stages of vascular development when vessels reorganize. The central hypothesis of this application is that Notch signaling regulates cellular and molecular processes essential for micro vascular differentiation and stability. It is well known that basement membrane thickening and pericyte loss are early changes associated with the development of diabetic retinopathy and we speculate that these events disrupt of Notch signaling. To assess the role of Notch signaling between endothelial cells and pericytes in the retinal microvasculature we propose the following aims. (i) To determine the expression of Notch 1 and Notch 3 and its ligands, Jagged 1 and Delta 1, in the adult retinal vasculature as well as in retinal vasculature of tissue from mice and humans with diabetic microangiopathy. The distribution of Notch pathway elements in the adult will be systematically examined using reporter mice and immunohistology. Expression of these signaling components will also be assessed in the retinal vasculature of mice with streptozotocin-induced diabetes and in tissues of human with background retinopathy. (ii) To assess the role of cell autonomous Notch signaling on pericyte stability/function in the adult. Mural cell-specific knockout and over expression strategies will be used to determine the role of Notch signaling in stabilization of the retinal vasculature. (iii) To assess the role of Notch signaling in EC-pericyte interactions in the adult. A co-culture model will be used to examine the role of Notch signaling in endothelial and mural cell function and gene expression. Mouse models described above will be used to examine how misregulation of Notch signaling in mural cells affect endothelial cell stability and function. A transgenic model in which diphtheria toxin is used to selectively delete pericytes will serve as a positive control. The proposed studies, which will examine the molecules involved in signaling between EC and pericytes, are critical to our understanding of the pathogenic process and to the development of effective means for prevention and treatment.